Orphan opioid receptor and recombinant materials for its production

ABSTRACT

Genes encoding opioid receptors (including opioid-like receptor (ORL) proteins) can be retrieved from vertebrate libraries using the murine probe disclosed herein under low-stringency conditions. The DNA sequence shown in FIG.  5  or its complement can be used to obtain the human delta, kappa and mu genes as well as the murine mu gene and human ORL-1. The probe provided encodes the murine delta opioid receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of Attorney Docket No.22000-20526.21 filed Mar. 13, 1995; which is a continuation-in-part ofU.S. Ser. No. 08/387,707 filed Feb. 13, 1995 (the National Phaseapplication of PCT US 93/07665 filed Aug. 13, 1993) which is acontinuation-in-part of U.S. Ser. No. 07/929,200 filed Aug. 13, 1992.The contents of these applications are incorporated herein by reference.

[0002] This invention was made with Government support under Grant No.DA05010 awarded by the Alcohol, Drug Abuse and Mental HealthAdministration. The Government has certain rights in the invention.

TECHNICAL FIELD

[0003] The invention relates to substances involved in vertebratenervous systems, and in particular to the opioid receptors andreceptor-like proteins (also referred to as opioid receptors herein) andactivities mediated thereby. Accordingly, the invention concernsrecombinant materials useful for the production of opioid receptors, thereceptor as a diagnostic tool, therapeutic and diagnostic compositionsrelevant to the receptor, and methods of using the receptor to screenfor drugs that modulate the activity of the receptor.

BACKGROUND ART

[0004] The term “opioid” generically refers to all drugs, natural andsynthetic, that have morphine-like actions. Formerly, the term “opiate”was used to designate drugs derived from opium, e.g., morphine, codeine,and many semi-synthetic congeners of morphine. After the isolation ofpeptide compounds with morphine-like actions, the term opioid wasintroduced to refer generically to all drugs with morphine-like actions.Included among opioids are various peptides that exhibit morphine-likeactivity, such as endorphins, enkephalins and dynorphins. However, somesources have continued to use the term “opiate” in a generic sense, andin such contexts, opiate and opioid are interchangeable. Additionally,the term opioid has been used to refer to antagonists of morphine-likedrugs as well as to characterize receptors or binding sites that combinewith such agents.

[0005] Opioids are generally employed as analgesics, but they may havemany other pharmacological effects as well. Morphine and related opioidsproduce their major effects on the central nervous and digestivesystems. The effects are diverse, including analgesia, drowsiness, moodchanges, respiratory depression, dizziness, mental clouding, dysphoria,pruritus, increased pressure in the biliary tract, decreasedgastrointestinal motility, nausea, vomiting, and alterations of theendocrine and autonomic nervous systems.

[0006] A significant feature of the analgesia produced by opioids isthat it occurs without loss of consciousness. When therapeutic doses ofmorphine are given to patients with pain, they report that the pain isless intense, less discomforting, or entirely gone. In addition toexperiencing relief of distress, some patients experience euphoria.However, when morphine in a selected pain-relieving dose is given to apain-free individual, the experience is not always pleasant; nausea iscommon, and vomiting may also occur. Drowsiness, inability toconcentrate, difficulty in mentation, apathy, lessened physicalactivity, reduced visual acuity, and lethargy may ensue.

[0007] The development of tolerance and physical dependence withrepeated use is a characteristic feature of all opioid drugs, and thepossibility of developing psychological dependence on the effect ofthese drugs is a major limitation for their clinical use. There isevidence that phosphorylation may be associated with tolerance inselected cell populations (Louie, A. et al. Biochem Biophys Res Comm(1988) 152:1369-75).

[0008] Acute opioid poisoning may result from clinical overdosage,accidental overdosage, or attempted suicide. In a clinical setting, thetriad of coma, pinpoint pupils, and depressed respiration suggest opioidpoisoning. Mixed poisonings including agents such as barbiturates oralcohol may also contribute to the clinical picture of acute opioidpoisoning. In any scenario of opioid poisoning, treatment must beadministered promptly.

[0009] The opioids interact with what appear to be several closelyrelated receptors. Various inferences have been drawn from data thathave attempted to correlate pharmacologic effects with the interactionsof opioids with a particular constellation of opioid receptors (Goodmanand Gilman's, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th ed, pp.493-95 (MacMillan 1985)). For example, analgesia has been associatedwith mu and kappa receptors. Delta receptors are believed to be involvedin alterations of affective behavior, based primarily on thelocalization of these receptors in limbic regions of the brain.Additionally, activation, e.g., ligand binding with stimulation offurther receptor-mediated responses, of delta opioid receptors isbelieved to inhibit the release of other neurotransmitters. The pathwayscontaining relatively high populations of delta opioid receptor aresimilar to the pathways implicated to be involved in Huntington'sdisease. Accordingly, it is postulated that Huntington's disease maycorrelate with some effect on delta opioid receptors.

[0010] Two distinct classes of opioid molecules can bind opioidreceptors: the opioid peptides (e.g., the enkephalins, dynorphins, andendorphins) and the alkaloid opiates (e.g., morphine, etorphine,diprenorphine and naloxone). Subsequent to the initial demonstration ofopiate binding sites (Pert, C. B. and Snyder, S. H., Science (1973)179:1011-1014), the differential pharmacological and physiologicaleffects of both opioid peptide analogues and alkaloid opiates served todelineate multiple opioid receptors. Accordingly, three anatomically andpharmacologically distinct opioid receptor types have been described:delta, kappa and mu. Furthermore, each type is believed to havesub-types (Wollemann, M., J Neurochem (1990) 54:1095-1101; Lord, J. A.,et al., Nature (1977) 267:495-499).

[0011] All three of these opioid receptor types appear to share the samefunctional mechanisms at a cellular level. For example, the opioidreceptors cause inhibition of adenylate cyclase, and inhibition ofneurotransmitter release via both potassium channel activation andinhibition of Ca²⁺ channels (Evans, C. J., In: Biological Basis ofSubstance Abuse, S. G. Korenman & J. D. Barchas, eds., Oxford UniversityPress (in press); North, A. R., et al., Proc Natl Acad Sci USA (1990)87:7025-29; Gross, R. A., et al., Proc Natl Acad Sci USA (1990)87:7025-29; Sharma, S. K., et al., Proc Natl Acad Sci USA (1975)72:3092-96). Although the functional mechanisms are the same, thebehavioral manifestations of receptor-selective drugs differ greatly(Gilbert, P. E. & Martin, W. R., J Pharmacol Exp Ther (1976) 198:66-82).Such differences may be attributable in part to the anatomical locationof the different receptors.

[0012] Delta receptors have a more discrete distribution within themammalian CNS than either mu or kappa receptors, with highconcentrations in the amygdaloid complex, striatum, substantia nigra,olfactory bulb, olfactory tubercles, hippocampal formation, and thecerebral cortex (Mansour, A., et al., Trends in Neurosci (1988)11:308-14). The rat cerebellum is remarkably devoid of opioid receptorsincluding delta opioid receptors.

[0013] Several opioid molecules are known to selectively orpreferentially bind delta receptors. Of the vertebrate endogenousopioids, the enkephalins, particularly met-enkephalin andleu-enkephalin, appear to possess the highest affinity for deltareceptors, although the enkephalins also have high affinity for mureceptors. Additionally, the deltorphans, peptides isolated from frogskin, comprise a family of opioid peptides that have high affinity andselectivity for delta receptors (Erspamer, V., et al., Proc Natl AcadSci USA (1989) 86:5188-92).

[0014] A number of synthetic enkephalin analogues are also deltareceptor-selective including (D-Ser²) leucine enkephalin Thr (DSLET)(Garcel, G. et al. FEBS Lett (1980) 118:245-247) and (D-Pen², D-Pen⁵)enkephalin (DPDPE) Akiyama, K. et al., Proc Natl Acad Sci USA (1985)82:2543-2547).

[0015] Recently a number of other selective delta receptor ligands havebeen synthesized, and their bioactivities and binding characteristicssuggest the existence of more than one delta receptor subtype (Takemori,A. E., et al., Ann Rev Pharm Toxicol, (1992) 32:239-69; Negri, L., etal., Eur J Pharmacol (1991) 196:355-335; Sofuoglu, M., et al.,Pharmacologist (1990) 32:151).

[0016] Although the synthetic pentapeptide 2dAla, 5dLeu enkephalin(DADLE) was considered to be delta-selective, it also binds equally wellto mu receptors. The synthetic peptideD-Ala²-N-Me-Phe⁴-Gly-ol⁵-enkephalin (DAGO) has been found to be aselective ligand for mu-receptors.

[0017] The existence of multiple delta opioid receptors has been impliednot only from the pharmacological studies addressed above, but also frommolecular weight estimates obtained by use of irreversible affinityligands. Molecular weights for the delta opioid receptor that range from30 kDa to 60 kda (Evans, C. J., supra; Evans, C. J. et al., Science(1992) 258:1952-1955, which document corresponds to the disclosure ofthe priority document of the present application; Bochet, P. et al., MolPharmacol (1988) 34:436-43). The various receptor sizes may representalternative splice products, although this has not been established.

[0018] Many studies of the delta opioid receptor have been performedwith the neuroblastoma/glioma cell line NG108-15, which was generated byfusion of the rat glial cell line (C6BU-1) and the mouse neuroblastomacell line (N18-TG2) (Klee, W. A. and Nirenberg, M. A., Proc Natl AcadSci USA (1974) 71:3474-3477). The rat glial cell line expressesessentially no delta opioid receptors, whereas the mouse neuroblastomacell line expresses low amounts of the receptor. Thus, it has beensuggested that the delta receptor in the NG108-15 cells is of mousechromosomal origin (Law, Mol Pharm (1982) 21:438-91). Each NG108-15 cellis estimated to express approximately 300,000 delta-receptors. Onlydelta-type opioid receptors are expressed, although it is not knownwhether these represent more than a single subtype. Thus, the NG108-15cell line has served to provide considerable insight into the bindingcharacterization of opioid receptors, particularly delta opioidreceptors. However, the NG108-15 cell line is a cancer-hybrid and maynot be completely representative of the delta receptor in endogenousneurons due to the unique cellular environment in the hybrid cells.

[0019] An extensive literature has argued that the opioid receptors arecoupled to G-proteins (see, e.g., Schofield, P. R., et al., EMBO J(1989) 8:489-95), and are thus members of the family of G-proteincoupled receptors. G-proteins are guanine nucleotide binding proteinsthat couple the extracellular signals received by cell surface receptorsto various intracellular second messenger systems. Identified members ofthe G-protein-coupled family share a number of structural features, themost highly conserved being seven apparent membrane-spanning regions,which are highly homologous among the members of this family (Strosberg,A. D., Eur J Biochem (1991) 196:1-10). Evidence that the opioidreceptors are members of this family includes the stimulation of GTPaseactivity by opioids, the observation that GTP analogues dramaticallyeffect opioid and opiate agonist binding, and the observation thatpertussis toxin (which by ADP ribosylation selectively inactivates boththe Gi and Go subfamilies of G-proteins) blocks opioid receptor couplingto adenylate cyclase and to K⁺ and Ca²⁺ channels (Evans, C. J., supra).

[0020] The members of the G-protein-coupled receptor family exhibit arange of characteristics. Many of the G-protein-coupled receptors, e.g.,the somatostatin receptor and the angiotensin receptor, have a singleexon that encodes the entire protein coding region (Strosberg supra;Langord, K., et al., Biochem Biophys Res Comm (1992) 138:1025-1032).However, other receptors, such as substance P receptor and the dopamineD-2 receptor, contain the protein coding region. The D-2 receptor isparticularly interesting in that alternate splicing of the gene givesrise to different transcribed products (i.e., receptors) (Evans, C. J.,supra; Strosberg, supra). Interestingly, somatostatin ligands arereported to bind to opioid receptors (Terenius, L., Eur J Pharmacol(1976) 38:211; Mulder, A. H., et al., Eur J Pharmacol (1991) 205:1-6)and, furthermore, to have similar molecular mechanisms (Tsunoo, A., etal., Proc Natl Acad Sci USA (1986) 83:9832-9836).

[0021] In previous efforts to describe and purify opioid receptors, twoclones have been described that were hypothesized either to encode aportion of or entire opioid receptors. The first clone, which encodesthe opiate binding protein OBCAM (Schofield et al., supra), was obtainedby utilizing a probe designed from an amino acid sequence of a proteinpurified on a morphine affinity column. OBCAM lacks any membranespanning domains but does have a C-terminal domain that ischaracteristic of attachment of the protein to the membrane by aphosphatidylinositol (PI) linkage. This feature, which is shared bymembers of the immunoglobulin superfamily, is not common to the familyof receptors coupled to G-proteins. Thus, it has been proposed thatOBCAM is part of a receptor complex along with other components that arecoupled to G-proteins (Schofield et al., supra). At present, however,there is no direct evidence for such a complex.

[0022] A second proposed opioid receptor clone was obtained in an effortto clone a receptor that binds kappa opioid receptor ligands (Xie, G.X., Proc Natl Acad Sci USA (1992) 89:4124-4128). A DNA molecule encodinga G-coupled receptor from a placental cDNA library was isolated. Thisreceptor has an extremely high homology with the neurokinin B receptor(84w identical throughout the proposed protein sequence). When thisclone was expressed in COS cells, it displayed opioid peptidedisplaceable binding of ³H-bremazocine (an opiate ligand with highaffinity for kappa receptors). However, the low affinity of thisreceptor for ³H-bremazocine, and the lack of appropriate selectivitysince this receptor (binding both mu and delta ligands) made it doubtfulthat this cloned molecule is actually an opioid receptor.

[0023] Furthermore, characterization of opioid receptor proteins hasproven difficult because of their instability once solubilized from themembrane; purified delta opioid receptors have not been isolated. Theprevious estimates of opioid receptor molecular weights ranging from 30kDa to 60 Kda further reflect the difficulty in isolating andcharacterizing these proteins.

[0024] Recently, DNA encoding the murine kappa and delta opioidreceptors from mouse brain was reported by Yasuda, K. et al. Proc NatlAcad Sci USA (1993) 90:6736-6740. The sequence of the clones indicatedthe presence of the expected seven transmembrane regions. In addition,Chen, Y. et al. in a soon-to-be-published manuscript in MolecularPharmacology (1993) report the “molecular cloning and functionalexpression of a mu opioid receptor from rat brain”. In fact, the rat mureceptor was cloned using the present inventors' DOR-1 clone, whichlends enabling support to the present invention disclosed below. Themouse delta opioid receptor was disclosed as having been cloned(Kieffer, B. J. et al., Proc Natl Acad Sci USA (1992) 89:12048-12052(December issue) after the filing date of the priority document of thepresent application. However, the sequence reported therein differs fromthe sequence reported by the present inventors for the mouse deltareceptor (Evans et al., 1992, supra; this disclosure).

[0025] In addition to the opioid receptors which respond to specifiedagonists, the delta, kappa and mu opioid receptors, additional forms ofthese proteins, commonly called opioid receptor-like (ORL) proteins havebeen obtained using the methods described herein. Using these methods,two human ORL protein-encoding cDNAs were obtained from a human brainstem cDNA library. One of these clones is equivalent to that isolated byO'Dowd, B. F. et al. Gene (1993) 136:355-360; the other, ORL-1, isidentical to that reported by Mollereau, C. et al. FEBS Lett (1994)341:33-38. A preliminary report of the present work appeared inRegulatory Peptides (1994) 54:143-144 and is incorporated herein byreference.

DISCLOSURE OF THE INVENTION

[0026] The present invention provides recombinant nucleic acid moleculeswhich encode the murine delta opioid receptor, as well as recombinantnucleic acid molecules which can be retrieved using low-stringencyhybridization to this disclosed DNA. Thus, the invention provides genesencoding the delta, kappa and mu receptors, representing opioidreceptors generally, including ORL proteins, of any species containinggenes encoding such receptors or ORL proteins sufficiently homologous tohybridize under low-stringency conditions described herein.

[0027] As used herein, “opioid receptors” includes not only thepreviously identified delta, kappa and mu receptors, but also additionalreceptor-like proteins, represented by, for example, ORL-1 thathybridize under the low-stringency conditions described to the murineDOR clone set forth herein, and which have opioid receptorcharacteristics including seven putative transmembrane regions, andability to couple with guanine nucleotide-binding regulatory proteins (Gproteins) to inhibit adenylyl cyclase and/or calcium channels or tostimulate potassium channels. Thus, when the word “opioid receptor” isused hereinbelow, this term is intended to include this entire genus.

[0028] Thus, in one aspect, the invention is directed to recombinantnucleic acid molecules and methods for the production of an opioidreceptor wherein the opioid receptor is encoded by a gene whichhybridizes under-low-stringency to the nucleotide sequence of FIG. 5 orto its complement. By “low-stringency” is meant 50% formamide/6× SSC,overnight at 37° C. for the hybridization, followed by washes at 2× SSC0.1% SDS at room temperature or 50% formamide/6× SSC at 37° C. withwashes of 1× SSC/0.1% SDS at 37° C.

[0029] Also provided are expression systems comprising the nucleic acidmolecules described above. The receptor can be recombinantly producedusing these expression systems and host cells modified to contain them.

[0030] Especially useful are vertebrate cells which express the opioidreceptor gene so that the opioid receptor protein is displayed at thesurface of the cells. These cells offer means to screen native andsynthetic candidate agonists and antagonists for the opioid receptors.

[0031] In still other aspects, the invention is directed to methods toscreen candidate agonists and/or antagonists acting at opioid receptorsusing the recombinant transformed cells of the invention. Such assaysinclude (1) binding assays using competition with ligands known to bindopioid receptors, (2) agonist assays which analyze activation of thesecondary pathways associated with opioid receptor activation in thetransformed cells, and (3) assays which evaluate the effect on bindingof the candidate to the receptor by the presence or absence of sodiumion and GTP. Antagonist assays include the combination of the ability ofthe candidate to bind the receptor while failing to effect furtheractivation, and, more importantly, competition with a known agonist.

[0032] Still another aspect of the invention is provision of antibodycompositions which are immunoreactive with the opioid receptor proteins.Such antibodies are useful, for example, in purification of thereceptors after solubilization or after recombinant production thereof.

[0033] In still other aspects, the invention is directed to probesuseful for the identification of DNA which encodes, related opioidreceptors in various species or different types and subtypes of opioidreceptors.

[0034] Accordingly, an object of the present invention is to provide anisolated and purified form of a DNA sequence encoding an opioidreceptor, which is useful as a probe as well as in the production of thereceptor.

[0035] Another object is to provide a recombinantly, produced DNAsequence encoding an opioid receptor.

[0036] Another object is to produce an antisense sequences correspondingto known sense sequences encoding the opioid receptors.

[0037] Another object of the invention is to provide a DNA constructcomprised of a control sequence operatively linked to a DNA sequencewhich encodes an opioid receptor and to provide recombinant host cellsmodified to contain the DNA construct.

[0038] Another object is to isolate, clone and characterize, fromvarious vertebrate species, DNA sequences encoding the various relatedreceptors, by hybridization of the DNA derived from such species with anative DNA sequence encoding the opioid receptor of the invention.

[0039] An advantage of the present invention is that opioidreceptor-encoding DNA sequences can be expressed at the surface of hostcells which can conveniently be used to screen drugs for their abilityto interact with and/or bind to the receptors.

[0040] These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 depicts a comparison of binding of ³H-diprenorphine(saturation curves) between NG108-15 cells and COS cells three daysfollowing transfection (by electroporation) of each with DOR-1 in theCDM-8 vector. Specific opioid binding was undetectable in nontransfectedCOS cells or COS cells transfected with plasmid alone.

[0042]FIG. 2 depicts displacement curves of 5 nM ³H-diprenorphine fromCOS cell membranes of cells transfected with DOR-1. ³H-diprenorphine wasdisplaced by diprenorphine, etorphine, morphine and levorphanol, but notby dextrorphan (the non-opiate active optical isomer of levorphanol).

[0043]FIG. 3 depicts displacement curves of 5 nm ³H-diprenorphine fromCOS cell membranes of cells transfected with DOR-1. ³H-diprenorphine wasdisplaced by DPDPE and DSLET, which are delta-selective agonists, byDADLE, a high affinity ligand for mu and delta receptors, and bydynorphin 1-17, a kappa-preferring ligand. ³H-diprenorphine was notdisplaced by DAGO, a mu-selective ligand.

[0044]FIG. 4 depicts the results of a Northern analysis of mRNA fromNG108-15 cells and cells from various rat brain regions.

[0045]FIG. 5 shows the nucleotide sequence and the deduced amino acidsequence of the DOR-1 clone.

[0046]FIG. 6 depicts the deduced amino acid sequence of DOR-1, comparedwith the rat somatostatin receptor. Consensus glycosylation sitespredicted to fall in extracellular domains are indicated by an asterisk.Potential protein kinase C sites are listed in Example 5. The sevenpredicted membrane spanning regions (underlined) are predicted based onthe hydrophobicity profile and published predictions (MacVector softwareprogram (IBI); T. Hopp, and K. Woods, Proc Natl Acad Sci USA (1981)78:3842-3828). For sequencing, the cDNA insert was subcloned intopBluescript and both strands were sequenced from single-stranded DNAusing Sequenase and Taq cycle sequencing. For ambiguities due tocompressions 7-deaza-dGTP replaced dGTP in the sequencing reactions andthe products were resolved on formamide gels.

[0047]FIG. 7 depicts a Southern blot of radiolabeled DOR-1 cDNA probehybridized at high stringency to NG108-15, mouse, rat and human DNA cutwith BamHI.

[0048]FIG. 8a shows a partial nucleotide sequence of the human deltaopioid receptor genomic clone H3 (also designated human DORa or hDORa).

[0049]FIG. 8b shows a partial nucleotide sequence of the human kappaopioid receptor genomic clone H14 (also designated human KORa or hKORa).

[0050]FIG. 8c shows a partial nucleotide sequence of the human mu opioidreceptor genomic clone H20 (also designated human MORa or hMORa).

[0051]FIG. 8d shows the nucleotide sequence of the CACACA repeat nearthe H20 DNA.

[0052]FIG. 9 shows the nucleotide sequence of the murine mu-receptorclone DOR-2 also named mMOR-1 or mMOR-1α.

[0053]FIG. 10 shows the homology of various receptor amino acidsequences.

[0054]FIG. 11 shows the complete DNA sequence of the cDNA retrieved froma human brain stem cDNA library and comprising a nucleotide sequenceencoding the opioid receptor ORL-1. This cDNA encodes a 370-amino acidopioid receptor protein.

[0055]FIG. 12 shows a comparison of ORL-1 and ORL-2 amino acid sequenceswith human and murine delta, kappa, and mu receptors. ORL-1 is a proteinof 370 amino acids and is compared with human mu opioid receptor (hMOR),human delta opioid receptor (hDOR) and murine kappa opioid receptor(mKOR).

MODES OF CARRYING OUT THE INVENTION

[0056] The invention provides DNA encoding mammalian opioid receptorprotein and additional recombinant nucleic acids, expression vectors andmethods useful for the production of these proteins. In addition,eucaryotic cells, such as COS cells, transformed with the recombinantmolecules of the invention so as to express opioid receptor proteins attheir surface are useful in screening assays to identify candidateopioid agonists and antagonists. In addition, antibodies may be raisedto the recombinantly produced opioid receptor proteins. These antibodiesare useful in immunoassays for said protein and in affinity purificationthereof.

[0057] Recombinant Opioid Receptor

[0058] Illustrated hereinbelow is the obtention of a cDNA encoding amurine delta opioid receptor. The complete DNA sequence of the cDNA, andthe amino acid sequence encoded thereby, are set forth herein in FIG. 5.The availability of this cDNA permits the retrieval of the correspondingopioid receptor-encoding DNA from other vertebrate species. Accordingly,the present invention places within the possession of the art,recombinant molecules and methods for the production of cells expressingopioid receptors of various types and of various vertebrate species.Thus, the cDNA of FIG. 5, or a portion thereof, may be used as a probeto identify that portion of vertebrate genomic DNA or cDNA which encodesan opioid receptor protein. Illustrative methods used to prepare agenomic library and identify the opioid receptor-encoding genes aredescribed for convenience hereinbelow. Also exemplified as illustratingthe method of the invention is the retrieval of human ORL-1 from a brainstem cDNA library.

[0059] The DOR-1 clone described in FIG. 5 is a cDNA clone correspondingto the murine delta opioid receptor. The present inventors found, anddescribe herein, that screening of a human genomic library underconditions of low stringency results in the recovery of DNA encoding allthree types of human opioid receptors. Similarly, a murine genomic clonewas obtained. In addition, a cDNA clone was obtained from a mouse brainlibrary encoding the murine mu opioid receptor. Thus, either cDNAlibraries from appropriate sources, such as brain, or genomic libraries,are fruitful sources or substrates for obtaining the DNA of the presentinvention and the corresponding recombinant materials. The invention isthus directed to DNA encoding an opioid receptor of a vertebrate,wherein the opioid receptor is encoded by a nucleotide sequence whichhybridizes under conditions of low stringency to the nucleotide sequenceshown in FIG. 5 or to its complement.

[0060] In the alternative, the DNA of FIG. 5 or a portion thereof may beused to identify specific tissues or cells which express opioid receptorprotein by analyzing the mRNA, for example, using Northern blottechniques. Those tissues which are identified as containing mRNAencoding opioid receptor protein using the probes of the invention arethen suitable sources for preparation of cDNA libraries which mayfurther be probed using the cDNA described hereinbelow.

[0061] The DNA encoding the various vertebrate opioid receptor proteins,obtained in general as set forth above, according to the standardtechniques described hereinbelow, can be used to produce cells whichexpress the opioid receptor at their surface; such cells are typicallyeucaryotic cells, in particular, mammalian cells such as COS cells orCHO cells. Suitable expression systems in eucaryotic cells for suchproduction are described hereinbelow. The opioid receptor proteins mayalso be produced in procaryotes or in alternative eucaryotic expressionsystems for production of the protein per se. The DNA encoding theprotein may be ligated into expression vectors preceded by signalsequences to effect its secretion, or may be produced intracellularly,as well as at the cell surface, depending on the choice of expressionsystem and host. If desired, the opioid receptor protein thusrecombinantly produced may be purified using suitable means of proteinpurification, and, in particular, by affinity purification usingantibodies or fragments thereof immunospecific for the opioid receptorprotein.

[0062] The reader is reminded that the term “opioid receptor” as usedherein includes not only the conventional delta, kappa and mu opioidreceptors, but also opioid receptor-like proteins which interact with Gproteins in a similar manner. These receptor-like proteins are useful inanalogous ways, and offer additional screening tools for candidatecompounds that affect the central nervous system. They are thus usefulfor the same purposes as the “conventional” receptors.

[0063] Screening for Opioid Agonists and Antagonists Using RecombinantCells

[0064] The ability of a candidate compound to act as an opioid agonistor antagonist may be assessed using the recombinant cells of theinvention in a variety of ways. To exhibit either agonist or antagonistactivity, the candidate compound must bind to the opioid receptor. Thus,to assess the ability of the candidate to bind, either a direct orindirect binding assay may be used. For a direct binding assay, thecandidate binding compound is itself detectably labeled, such as with aradioisotope or fluorescent label, and binding to the recombinant cells.of the invention is assessed by comparing the acquisition of label bythe recombinant cells to the acquisition of label by correspondinguntransformed (control) cells.

[0065] More convenient, however, is the use of a competitive assaywherein the candidate compound competes for binding to the recombinantcells of the invention with a detectably labeled form of an opioidligand known to bind to the receptor. Such ligands are themselveslabeled using radioisotopes or fluorescent moieties, for example. Aparticularly suitable opioid known to bind to this receptor isdiprenorphine. A typical protocol for such an assay is as follows:

[0066] In general, about 10⁶ recombinant cells are incubated insuspension in 1.0 ml of Kreb's Ringer Hepes Buffer (KRHB) at pH 7.4, 37°C. for 20 min with ³H-diprenorphine. Nonspecific binding is determinedby the addition of 400 nM diprenorphine in the binding mixtures. Variousconcentrations of candidate compounds are added to the reactionmixtures. The incubations are terminated by collecting the cells onWhatman GF-B filters, with removal of excess radioactivity by washingthe filters three times with 5 ml of KRHB at 0° C. After incubating at20° C. overnight in 5 ml of scintillation fluid, such as Liquiscint(National Diagnostics, Somerville, N.J.), the radioactivity on thefilters is determined by liquid scintillation counting.

[0067] The K_(d) (dissociation constant) values for the candidate opiateligands can be determined from the IC50 value (“inhibitoryconcentration₅₀” means the concentration of candidate ligand thatresults in a 50%. decrease in binding of labeled diprenorphine).

[0068] The effects of sodium and GTP on the binding of ligands to therecombinantly expressed receptors can be used to distinguish agonistfrom antagonist activities. If the binding of a candidate compound issensitive to Na⁺ and GTP, it is more likely to be an agonist than anantagonist, since the functional coupling of opioid receptors to secondmessenger molecules such as adenylate cyclase requires the presence ofboth sodium and GTP (Blume et al., Proc Natl Acad Sci USA (1979)73:26-35). Furthermore, sodium, GTP, and GTP analogues have been shownto effect the binding of opioids and opioid agonists to opioid receptors(Blume, Life Sci (1978) 22:1843-52). Since opioid antagonists do notexhibit binding that is sensitive to guanine nucleotides and sodium,this effect is used as a method for distinguishing agonists fromantagonists using binding assays.

[0069] In addition, agonist activity can directly be assessed by thefunctional result within the cell. For example, it is known that thebinding of opioid agonists inhibits cAMP formation, inhibits potassiumchannel activation, inhibits calcium channel activation, and stimulatesGTPase. Assessment of these activities in response to a candidatecompound is diagnostic of agonist activity. In addition, the ability ofa compound to interfere with the activating activity of a known agonistsuch as etorphine effectively classifies it as an antagonist.

[0070] In one typical assay, the measurement of cAMP levels in cellsexpressing opioid receptors is carried out by determining the amount of³H-cAMP formed from intracellular ATP pools prelabeled with ³H-adenine(Law et al., supra). Thus, cAMP formation assays are carried out with0.5×10⁶ cells/0.5 ml of KRHB at pH 7.4, incubated at 37° C. for 20minutes. After addition of the internal standard ³²P-cAMP, theradioactive cAMP is separated from other ³H-labeled nucleotides by knowndouble-column chromatographic methods. The opiate agonists' ability toinhibit cAMP accumulation is then determined as described by Law et al.(supra).

[0071] The potency of a candidate opiate antagonist can be determined bymeasuring the ability of etorphine to inhibit cyclic AMP accumulation inthe presence and in the absence of known amounts of the candidateantagonist. The inhibition constant (K_(i)) of an antagonist can then becalculated from the equation for competitive inhibitors.

[0072] An interesting feature of screening assays using the prior artNG108-15 cells is that the agonist adenylate cyclase inhibition functionapparently does not require binding of all receptors on these cells.Thus, the K_(d) and K_(i) values for the opioid ligands differed whenusing these cells.

[0073] The foregoing assays, as described above, performed on therecombinantly transformed cells of the present invention, provide a moredirect and more convenient screen for candidate compounds having agonistand antagonist opioid receptor activity than that previously availablein the art. Furthermore, such assays are more sensitive since cells can,in accordance with the present invention, be engineered to express highlevels of the opioid receptor. Additionally, cells engineered inaccordance with the present invention will-circumvent the concern thatNG108-15 cells, due to their tumor cell background, have a cellularenvironment that artifactually affects opioid receptor expression.

[0074] The mu opioid encoding DNA described herein also offer a means tofollow inheritance patterns. DNA sequence polymorphisms frequently occurin the noncoding regions that surround genes. Polymorphisms areespecially frequent in repeat sequences such as CACACA which often showdistinct polymorphisms in the number of repeats that are present indifferent individuals. These polymorphisms offer a marker by which tofollow the inheritance of the gene among family members. The inheritanceof a gene (such as MORa) or its human counterpart can be followed bypolymerase chain reaction (PCR) amplification of the region surroundingthe CACACA polymorphism and analyzing the resulting products. This wouldbe a useful diagnostic marker for the mu opioid receptor gene.

[0075] Methods to Prepare Opioid Receptor Protein or Portions Thereof

[0076] The present invention provides the amino acid sequence of amurine opioid receptor; similarly, the availability of the cDNA of theinvention places within possession of the art corresponding vertebrateopioid receptors whose amino acid sequence may also be determined bystandard methods. As the amino acid sequences of such opioid receptorsare known, or determinable, in addition to purification of such receptorprotein from native sources, recombinant production or synthetic peptidemethodology may also be employed for producing the receptor protein orpeptide.

[0077] The opioid receptor or portions thereof can thus also be preparedusing standard solid phase (or solution phase) peptide synthesismethods, as is known in the art. In addition, the DNA encoding thesepeptides may be synthesized using commercially available oligonucleotidesynthesis instrumentation for production of the protein in the mannerset forth above. Production using solid phase peptide synthesis is, ofcourse, required if amino acids not encoded by the gene are to beincluded.

[0078] The nomenclature used to describe the peptides and proteins ofthe invention follows the conventional practice where the N-terminalamino group is assumed to be to the left and the carboxy group to theright of each amino acid residue in the peptide. In the formulasrepresenting selected specific embodiments of the present invention, theamino- and carboxy-terminal groups, although often not specificallyshown, will be understood to be in the form they would assume atphysiological pH values, unless otherwise specified. Thus, theN-terminal NH3⁺ and C-terminal COO⁻ at physiological pH are understoodto be present though not necessarily specified and shown, either inspecific examples or in generic formulas. Free functional groups on theside chains of the amino acid residues may also be modified byglycosylation, phosphorylation, cysteine binding, amidation, acylationor other substitution, which can, for example, alter the physiological,biochemical, or biological properties of the compounds without affectingtheir activity within the meaning of the appended claims.

[0079] In the peptides shown, each gene-encoded residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the amino acid, in accordance withthe following conventional list: One-Letter Three-letter Amino AcidSymbol Symbol Alanine A Ala Arginine R Arg Asparagine N Asn Asparticacid D Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine GGly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K LysMethionine M Met Phenylalanine F Phe Proline P Pro Serine S SerThreonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

[0080] Nomenclature of Enkephalins

[0081] Enkephalins are either of two peptides having five residues withthe N-terminal residue numbered 1:

[0082] tyr-gly-gly-phe-xxx

[0083] 2 3 4 5

[0084] In “met enkephalin” the fifth residue is methionine:

[0085] tyr-gly-gly-phe-met

[0086] In “leu enkephalin” the 5th residue is leucine:

[0087] tyr-gly-gly-phe-leu

[0088] Enkephalin analogs can be made with (1) amino acid substitutions,(2) D-amino acid substitutions, and/or (3) additional amino acids. Thesite at which the substitution is made is noted at the beginning of thecompound name. For example, “(D-ala², D-leu⁵) enkephalin” means thatD-ala is present at the second position and D-leu is present at thefifth position:

[0089] tyr-[D-ala]-gly-phe-[D-leu]

[0090] One letter abbreviations can also be used. Thus, “(D-ser²) leuenkephalin” could be abbreviated as “DSLE.”Additional residues are notedas well. Thus, the addition of a threonine residue (to the sixthposition) of (D-ser²) leu enkephalin would be “(D-ser²) leu enkephalinthr” which could be abbreviated as “DSLET”:

[0091] tyr-[D-ser]-gly-phe-leu-thr

[0092] Antibodies

[0093] Antibodies immunoreactive with the opioid receptor protein orpeptide of the present invention can be obtained by immunization ofsuitable mammalian subjects with peptides, containing as antigenicregions those portions of the receptor intended to be targeted by theantibodies. Certain protein sequences have been determined to have ahigh antigenic potential. Such sequences are listed in antigenicindices, for example, MacVector software (I.B.I.) Thus, by determiningthe sequence of the opioid receptor protein and evaluating the sequencewith an antigenic index, probable antigenic sequences are located.

[0094] Antibodies are prepared by immunizing suitable mammalian hostsaccording to known immunization protocols using the peptide haptensalone, if they are of sufficient length, or, if desired, or if requiredto enhance immunogenicity, conjugated to suitable carriers. Methods forpreparing immunogenic conjugates with carriers such as BSA, KLH, orother carrier proteins are well known in the art. In some circumstances,direct conjugation using, for example, carbodiimide reagents may beeffective; in other instances linking reagents such as those supplied byPierce Chemical Co., Rockford, Ill., may be desirable to provideaccessibility to the hapten. The hapten peptides can be extended orinterspersed with cysteine residues, for example, to facilitate linkingto carrier. Administration of the immunogens is conducted generally byinjection over a suitable time period and with use of suitableadjuvants, as is generally understood in the art. During theimmunization schedule, titers of antibodies are taken to determineadequacy of antibody formation.

[0095] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions, useof monoclonal antibody (mAb) preparations is preferred. Immortalizedcell lines which secrete the desired mAbs may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired mAbs are screened byimmunoassay in which the antigen is the peptide hapten or is the opioidreceptor itself displayed on a recombinant host cell. When theappropriate immortalized cell culture secreting the desired mAb isidentified, the cells can be cultured either in vitro or byintraperitoneal injection into animals wherein the mAbs are produced inthe ascites fluid.

[0096] The desired mAbs are then recovered from the culture supernatantor from the ascites fluid. In addition to intact antibodies, fragmentsof the mAbs or of polyclonal antibodies which contain theantigen-binding portion can be used as antagonists. Use ofimmunologically reactive antigen binding fragments, such as the Fab,Fab′, of F(ab′)₂ fragments, is often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin molecule.

[0097] Standard Methods

[0098] The techniques for sequencing, cloning and expressing DNAsequences encoding the amino acid sequences corresponding to a opioidreceptor, e.g., polymerase chain reaction (PCR), synthesis ofoligonucleotides, probing a cDNA library, transforming cells,constructing vectors, preparing antisense oligonucleotide sequencesbased on known sense nucleotide sequences, extracting messenger RNA,preparing cDNA libraries, and the like are well-established in the art.Ordinarily skilled artisans are familiar with the standard resourcematerials, specific conditions and procedures. The following paragraphsare provided for convenience, it being understood that the invention islimited only by the appended claims.

RNA Preparation and Northern Blot

[0099] RNA preparation is as follows: The samples used for preparationof RNA are immediately frozen in liquid nitrogen and then stored untiluse at −80° C. The RNA is prepared by CsCl centrifugation (Ausubel etal., supra) using a modified homogenization buffer (Chirgwin et al.,Biochemistry (1979) 18:5294-5299). Poly(A⁺) RNA is selected by oligo(dT)chromatography (Aviv and Leder, Proc Natl Acad Sci USA (1972)69:1408-1412). RNA samples are stored at −80° C.

[0100] Analysis of gene expression and tissue distribution can beaccomplished using Northern blots with, e.g., radiolabeled probes. ThemRNA is size-separated using gel electrophoresis and then typically istransferred to a nylon membrane or to nitrocellulose and hybridized withradiolabeled probe. Presence of the hybridized probe is detected usingautoradiography.

[0101] Cloning

[0102] The cDNA sequences encoding the opioid receptor protein areobtained from a random-primed, size-selected cDNA library.

[0103] Alternatively, the cDNA sequences encoding opioid receptorprotein are obtained from a CDNA library prepared from mRNA isolatedfrom cells expressing the receptor protein in various organs such as thebrain, according to procedures described in Sambrook, J. et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1989.

[0104] The cDNA insert from the successful clone, excised with arestriction enzyme such as EcoRI, is then used as a probe of theoriginal cDNA library or other libraries (low stringency) to obtain theadditional clones containing inserts encoding other regions of theprotein that together or alone span the entire sequence of nucleotidescoding for the protein.

[0105] An additional procedure for obtaining cDNA sequences encoding theopioid receptor protein is PCR. PCR is used to amplify sequences from apooled cDNA library of reversed-transcribed RNA, using oligonucleotideprimers based on the transporter sequences already known.

[0106] Vector Construction

[0107] Construction of suitable vectors containing the desired codingand control sequences employs ligation and restriction techniques whichare well understood in the art (Young et al., Nature (1988)316:450-452). Double-stranded cDNA encoding opioid receptor protein issynthesized and prepared for insertion into a plasmid vector CDM8.Alternatively, vectors such as Bluescript² or Lambda ZAP² (Stratagene,San Diego, Calif.) or a vector from Clontech (Palo Alto, Calif.) can beused in accordance with standard procedures (Sambrook, J. et al.,supra).

[0108] Site specific DNA cleavage is performed by treating with thesuitable restriction enzyme, such as EcoRI, or more than one enzyme,under conditions which are generally understood in the art, and theparticulars of which are specified by the manufacturer of thesecommercially available restriction enzymes. See, e.g., New EnglandBiolabs, Product Catalog. In general, about 1 μg of DNA is cleaved byone unit of enzyme in about 20 μl of buffer solution; in the examplesherein, typically, an excess of restriction enzyme is used to ensurecomplete digestion of the DNA substrate. Incubation times of about oneto two hours at about 37° C. are workable, although variations can betolerated. After each incubation, protein is removed by extraction withphenol/chloroform, and can be followed by other extraction and thenucleic acid recovered from aqueous fractions by precipitation withethanol.

[0109] In vector construction employing “vector fragments”, the vectorfragment is commonly treated with bacterial alkaline phosphatase (BAP)or calf intestinal alkaline phosphatase (CIP) in order to remove the 5′phosphate and prevent religation of the vector. Digestions are conductedat pH 8 in approximately 150 mM Tris, in the presence of Na⁺ and Mg⁺⁺using about 1 unit of BAP or CIP per μg of vector at 60° C. or 37° C.,respectively, for about one hour. In order to recover the nucleic acidfragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors which have been double digested by additional restriction enzymedigestion of the unwanted fragments.

[0110] Ligations are performed in 15-50 μl volumes under the followingstandard conditions and temperatures: 20 mM Tris-HCl, pH 7.5, 10 mMMgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM to 50 mM NaCl, and either 40 μMATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 33-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). Intermolecular blunt end ligations (usuallyemploying a 10-30 fold molar excess of linkers) are performed at 1 μMtotal ends concentration. Correct ligations for vector construction areconfirmed according to the procedures of Young et al., supra.

[0111] cDNA Library Screening

[0112] cDNA libraries can be screened using reduced stringencyconditions as described by Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York(1990), or by using methods described in Sambrook et al. supra), or byusing a colony or plaque hybridization procedure with a fragment of theDOR-1 cDNA coding for opioid receptor protein.

[0113] Plaque hybridization is typically carried out as follows: Hostbacteria such as LE 392 (Stratagene) are grown overnight at 370 in LBBroth (Sambrook et al., supra), gently pelleted and resuspended in onehalf the original volume of 10 mM MgSO₄, 10 mM CaCl₂. After titration,an amount of the phage library containing approximately 50,000 plaqueforming units (pfu) is added to 300 μl of the host bacteria, incubatedat 370 for 15 minutes and plated onto NZYCM agar with 10 ml NZYCM topagarose. A total of a million plaques distributed on twenty 15 cm platesare screened. For colony screening, transfected bacteria are plated ontoLB broth plates with the appropriate antibiotics. After the plaques orcolonies have grown to 1 mm, the plates are chilled at 4° C. for atleast two hours, and then overlaid with duplicate nitrocellulosefilters, followed by denaturation of the filters in 0.5 M NaOH/1.5 MNaCl for five minutes and neutralization in 0.5 M Tris, pH 7.4/1.5 MNaCl for five minutes. The filters are then dried in air, baked at 80°C. for two hours, washed in 5× SSC/0.5% SDS at 68° C. for several hours,and prehybridized in 0.5 M NaPO₄, pH 7.2/1% BSA/1 mM EDTA/7% SDS/100μg/ml denatured salmon sperm DNA for more than 4 hours. Using the DOR-1cDNA (described herein) labeled by random priming as a probe, highstringency hybridization is carried out in the same solution at 68° C.,and the temperature is reduced to 50-60° C. for lower stringencyhybridization. After hybridization for 16-24 hours, the filters arewashed first in 40 mM NaPO4, pH 7.2/0.5% BSA/5% SDS/1 mM EDTA twice forone hour each, then in 40 mM NaPO₄, pH 7.2/1% BSA/1 mM EDTA for one houreach, both at the same temperature as the hybridization (Boulton et al.,Cell (1991) 65:663-675). The filters are then exposed to film with anenhancing screen at −70° C. for one day to one week.

[0114] Positive signals are then aligned to the plates, and thecorresponding positive phage is purified in subsequent rounds ofscreening, using the same conditions as in the primary screen. Purifiedphage clones are then used to prepare phage DNA for subcloning into aplasmid vector for sequence analysis. Tissue distribution of DNAcorresponding to the various independent clones is analyzed usingNorthern blots and in situ hybridization using standard methods.Function of the DNA is tested using expression in a heterologouseucaryotic expression system such as COS cells.

[0115] Expression of Opioid Receptor Protein

[0116] The nucleotide sequence encoding opioid receptor protein can beexpressed in a variety of systems. The cDNA can be excised by suitablerestriction enzymes and ligated into procaryotic or eucaryoticexpression vectors for such expression.

[0117] For example, as set forth below, the cDNA encoding the protein isexpressed in COS cells. To effect functional expression, the plasmidexpression vector CDM8 (Aruffo and Seed, Proc Natl Acad Sci USA (1987)84:8573-8577, provided by Drs. Aruffo and Seed (Harvard University,Boston, Mass.) was used. Alternatively, other suitable expressionvectors such as retroviral vectors can be used.

[0118] Procaryotic and preferably eucaryotic systems can be used toexpress the opioid receptor. Eucaryotic microbes, such as yeast, can beused as hosts for mass production of the opioid receptor protein.Laboratory strains of Saccharomyces cerevisiae, Baker's yeast, are usedmost, although a number of other strains are commonly available. Vectorsemploying, for example, the 2μ origin of replication (Broach, Meth Enz(1983) 101:307), or other yeast compatible origins of replications(e.g., Stinchcomb et al., Nature (1979) 282:39); Tschempe et al., Gene(1980) 10:157; and Clarke et al., Meth Enz (1983) 101:300) can be used.Control sequences for yeast vectors include promoters for the synthesisof glycolytic enzymes (Hess et al., J Adv Enzyme Reg (1968) 7:149;Holland et al., Biochemistry (1978) 17:4900). Additional promoters knownin the art include the promoter for 3-phosphoglycerate kinase (Hitzemanet al., J Biol Chem (1980) 255:2073), and those for other glycolyticenzymes. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and enzymesresponsible for maltose and galactose utilization. It is also believedterminator sequences are desirable at the 3′ end of the codingsequences. Such terminators are found in the 3′ untranslated regionfollowing the coding sequences in yeast-derived genes.

[0119] Alternatively, genes encoding opioid receptor protein areexpressed in eucaryotic host cell cultures derived from multicellularorganisms. (See, e.g., Tissue Cultures, Academic Press, Cruz andPatterson, eds, (1973)). These systems have the additional advantage ofthe ability to splice out introns, and thus can be used directly toexpress genomic fragments. Useful host cell lines include amphibianoocytes such as Xenopus oocytes, COS cells, VERO and HeLa cells, Chinesehamster ovary (CHO) cells, and insect cells such as SF9 cells.Expression vectors for such cells ordinarily include promoters andcontrol sequences compatible with mammalian cells such as, for example,the commonly used early and late promoters from baculovirus, vacciniavirus, Simian Virus 40 (SV40) (Fiers et al., Nature (1973) 273:113), orother viral promoters such as those derived from polyoma, Adenovirus 2,bovine papilloma virus, or avian sarcoma viruses. The controllablepromoter, hMTII (Karin et al., Nature (1982) 299:797-802) may also beused. General aspects of mammalian cell host system transformations havebeen described by Axel, U.S. Pat. No. 4,399,216. It now appears, that“enhancer” regions are important in optimizing expression; these are,generally, sequences found upstream or downstream of the promoter regionin non-coding DNA regions. Origins of replication can be obtained, ifneeded, from viral sources. However, integration into the chromosome isa common mechanism for DNA replication in eucaryotes.

[0120] If procaryotic systems are used, an intronless coding sequenceshould be used, along with suitable control sequences. The cDNA ofopioid receptor protein can be excised using suitable restrictionenzymes and ligated into procaryotic vectors along with suitable controlsequences for such expression.

[0121] Procaryotes most frequently are represented by various strains ofE. coli; however, other microbial species and strains may also be used.Commonly used procaryotic control sequences which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, including suchcommonly used promoters as the β-lactamase (penicillinase) and lactose(lac) promoter systems (Chang et al., Nature (1977) 198:1056) and thetryptophan (trp) promoter system (Goeddel et al., Nucl Acids Res (1980)8:4057) and the X derived PL promoter and N-gene ribosome binding site(Shimatake et al., Nature (1981) 292:128).

[0122] Depending on the host cell used, transformation is carried outusing standard techniques appropriate to such cells. The treatmentemploying calcium chloride, as described by Cohen, Proc Natl Acad SciUSA (1972) 69:2110 (1972) or by Sambrook et al. (supra), can be used forprocaryotes or other cells which contain substantial cell wall barriers.For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology (1978) 54:546,optionally as modified by Wigler et al., Cell (1979) 16:777-785, or byChen and Okayama, supra, can be used. Transformations into yeast can becarried out according to the method of Van Solingen et al., J Bact(1977) 130:946, or of Hsiao et al., Proc Natl Acad Sci USA (1979)76:3829.

[0123] Other representative transfection methods include viraltransfection, DEAE-dextran mediated transfection techniques, lysozymefusion or erythrocyte fusion, scraping, direct uptake, osmotic orsucrose shock, direct microinjection, indirect microinjection such asvia erythrocyte-mediated techniques,. and/or by subjecting host cells toelectric currents. The above list of transfection techniques is notconsidered to be exhaustive, as other procedures for introducing geneticinformation into cells will no doubt be developed.

[0124] Modulation of Expression by Antisense Sequences

[0125] Alternatively, antisense sequences may be inserted into cellsexpressing opioid receptors as a means to modulate functional expressionof the receptors encoded by sense oligonucleotides. The antisensesequences are prepared from known sense sequences (either DNA or RNA),by standard methods known in the art. Antisense sequences specific forthe opioid receptor gene or RNA transcript can be used to bind to orinactivate the oligonucleotides encoding the opioid receptor.

[0126] Terminology

[0127] As used herein, the singular forms “a”, “an” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, reference to “a receptor” includes mixtures of suchreceptors, reference to “an opioid” includes a plurality of and/ormixtures of such opioids and reference to “the host cell” includes aplurality of such cells of the same or similar type and so forth.

[0128] Unless defined otherwise all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The following examplesare intended to illustrate but not to limit the invention. Temperaturesare in ° C. and pressures at near atmospheric unless otherwisespecified.

Preparation of Mono ¹²⁵I-DADLE

[0129] DADLE (Peninsula Laboratories Inc.) was iodinated using theiodogen method (Maidment et al., in: MICRODIALYSIS IN THE NEUROSCIENCES,T. Robinson and J. Justice, eds., pp. 275-303 (Elsevier, 1991)). Bothmono- and di-iodinated forms are produced. It has been reported thatdi-iodo-DADLE does not bind opiate receptors, due to the di-iodinationof the tyrosine residue (Miller, R. J., et al., Life Sci (1978)22:379-88). Accordingly, mono-iodinated DADLE is preferred.Mono-¹²⁵I-DADLE is also preferred because it has extremely high specificactivity compared to DADLE labeled with other isotopes. Thus, exposuretimes on the order of days, rather than weeks or months can be used.

[0130] By employing a molar ratio of sodium iodide to peptide ofapproximately 1:100 when carrying out iodination, the yield of thepreferred mono-iodinated DADLE was increased. Additionally, to furtherenhance the yield of the mono-iodinated form, iodinated DADLE(containing both mono- and di-iodinated forms) was purified byreverse-phase HPLC (Maidment et al., supra).

[0131] Employing this procedure a single major radiolabeled peak of themono-iodinated DADLE separated from di-iodinated and non-iodinatedforms.

[0132] DADLE monolabeled with ¹²⁵I is crucial to successful screening.Radiolabeled ¹²⁵I-DADLE differs from DADLE in several importantparameters: size, hydrophobicity, and binding affinity (slightly lower).The purification of mono-iodinated from di-iodinated and non-iodinatedDADLE by the HPLC step yields a ligand with very high specific activity(approximately 2000 Ci/mmol). The specific activity of themono-iodinated form is approximately 100 times greater than thatobtained by using the unseparated mixture of mono-, di-, andnon-iodinated DADLE. Monolabeled ¹²⁵I-DADLE must be used is within a fewdays of its preparation.

EXAMPLE 1 Preparation of DOR-1

[0133] The NG108-15 cell line (available from Dr. Christopher Evans,UCLA) comprises a homogeneous and enriched source of delta opioidreceptors. Utilizing mRNA isolated from NG108-15, a random-primed,size-selected cDNA library was constructed in plasmid vector CDM8. ThecDNA library was amplified in bacteria. The cDNA library was transfectedinto COS-7 cells by electroporation. Transiently transfected COS lawnswere screened and selected with highly purified mono-¹²⁵I-2dAla, 5dLeuenkephalin (¹²⁵I-DADLE). Positive clones were identified by filmautoradiography, and plasmids from these cells were recovered andamplified in bacteria. Thereafter, the plasmids were re-transfected intoCOS cells. Following three cycles of such plasmid enrichment, individualclones were transfected and a pure clone was identified that bound¹²⁵I-DADLE.

[0134] A. Construction of the cDNA Library

[0135] RNA was prepared from NG108-15 cells by homogenization in 6 Mguanidinium isothiocyanate, followed by centrifugation through cesiumchloride (J. M. Chirgwin, et al., Biochemistry (1979) 18:5294). Poly-A⁺RNA was isolated by chromatography over oligo-dT-cellulose (H. Aviv andP. Leder, Proc Natl Acad Sci USA (1972) 69:1408). Using this RNA as atemplate, random hexamers were used to prime cDNA synthesis by avianmyeloblastosis virus reverse transcriptase (Life Sciences Inc.). Secondstrand synthesis was accomplished with RNase-H and E. coli DNApolymerase (U. Gubler and B. J. Hoffman, Gene (1983) 24:263). The endsof the cDNAs were rendered blunt with T4 DNA polymerase and BstXIlinkers were added. cDNA longer than 1.5 kb was selected byelectrophoresis through 5% acrylamide followed by electro-elution. The1.5 kb cDNA was ligated to the CDM8 vector (A. Aruffo and B. Seed,supra, and then transformed into MC-1061 bacteria by electroporation (W.J. Dower et al., Nucl Acids Res (1988) 16:6127). Accordingly, six poolsof plasmid DNA were prepared from the original cDNA library ofapproximately 2×10⁶ recombinants.

[0136] B. Plasmid Transfection by Electroporation and Expression in COScells

[0137] COS cells were grown at high density and were harvested intrypsin, then resuspended at 2×10⁷/ml in 1.2× RPMI containing 20% fetalcalf serum. These cells were then incubated for ten minutes at 4° C.with 20 μg recombinant plasmid DNA from the cDNA library describedabove, and then electroporated at 960 μF and 230 V in a 0.4 cm gapcuvette (BioRad). The cells were then incubated an additional tenminutes at 4° C., and then plated into Dulbecco's Modified Eagle'sMedium (DMEM) plus 10% fetal calf serum (FCS).

[0138] C. Screening of Transfected COS Cells

[0139] The transfected COS cells as obtained above were grown for threedays, then screened using radiolabeled mono ¹²⁵I-DADLE. Transfected COSlawns were washed with PBS, then incubated at room temperature with10-20 nM ¹²⁵I-DADLE in KHRB containing 1% BSA. After 1 hour, the plateswere washed rapidly several times with ice cold PBS then dried on icewith strong flow of forced cold air. Plates were exposed on DupontCronex film in cassettes at room temperature. Positive clones wereidentified by careful alignment of the film with the petri dish via lowpower microscopy.

[0140] DNA was removed from positive cells by solubilization in 0.1% SDSin TE containing 1 μg/μl tRNA delivered from a syringe attached to acapillary tube on a micromanipulator. Plasmids were purified from theextracted cells using the Hirt lysis procedure (Hirt, B., J Mol Biol(1967) 26:365-369), and electroporated into MC-1061 bacteria. Theplasmids were purified then retransfected into COS cells. Followingthree such enriching cycles, individual plasmid clones were transfectedinto COS cells yielding a single clone, named the DOR-1 clone.

EXAMPLE 2 Characterization of DOR-1

[0141] The DOR-1 clone initially was characterized by screening cellmembrane fractions, from cells expressing DOR-1, with the labelled DADLEit was found that binding of ¹²⁵I DADLE was displaced by nanomolarconcentrations of opiate alkaloids diprenorphine, morphine, etorphine,and by DADLE, DSLET and DPDPE. Dextrorphan (10 μM) did not displace the¹²⁵I DADLE, whereas its opioid-active enantiomer levorphanol diddisplace the radiolabeled DADLE. Additionally, the mu receptor-selectiveligand DAGO (5μM) did not displace the counts.

[0142] The DOR-1 clone was further characterized pharmacologically byassessing binding of ³H-diprenorphine to intact cells expressing theDOR-1 clone (FIG. 1), and by assessing displacement of ³H-diprenorphinefrom membrane fractions of such cells (FIGS. 2 and 3).

[0143] Binding assays were conducted on intact cells in KRHB, 1% BSA; oron membranes in 25 mM HEPES, 5 mM MgCl₂ pH 7.7. Cells were harvestedwith PBS containing 1 mM EDTA, washed 2× with PBS then resuspended inKHRB. Membranes prepared from the cells (Law P. Y. E et al., Mol Pharm(1983) 23:26-35) were used directly in the binding assay. Binding assayswere conducted in 96 well polypropylene cluster plates (Costar), at 4°C. in a total volume of 100 μl with an appropriate amount ofradiolabeled ligand. Following 1 hour of incubation, plates wereharvested on a Tomtec harvester and “B” type filtermats were counted ina Betaplate (Pharmacia) scintillation counter using Meltilex B/HS(Pharmacia) melt-on scintillator sheets.

[0144] Intact cells expressing DOR-1 were analyzed with the highaffinity opiate antagonist ³H-diprenorphine. Specific binding wasdefined by the counts displaced by 400 nM diprenorphine. FIG. 1 shows asaturation curve for ³H-diprenorphine for NG108-15 cells, and COS-7cells transfected with the delta opioid receptor clone. UntransfectedCOS cells, or COS cells transfected with plasmid having no insert showedno specific binding. Thus, the opioid binding of COS-DOR-1 cells wassimilar to that of NG108-15 cells.

[0145] Membranes prepared by standard methods from transfected COS-7cells were employed for a more extensive pharmacologicalcharacterization of the receptor encoded by the DOR-1 clone. Theaffinities for the following alkaloid opiates in competition for³H-diprenorphine are illustrated in FIG. 2: unlabeled diprenorphine, ahigh affinity antagonist for delta receptors; etorphine, a high affinityagonist for delta, mu and kappa receptors; levorphanol, a low affinityagonist for delta receptors; morphine, a low affinity agonist for deltareceptors and a high affinity agonist for mu receptors; and dextrorphan,a non-opiate active enantiomer of levorphanol which should not binddelta receptors.

[0146] As shown in FIG. 2, the displacement of ³H-diprenorphine, indecreasing order of affinity, was observed with diprenorphine,etorphine, levorphanol and morphine. As expected, ³H-diprenorphine wasnot displaced by dextrorphan.

[0147] The affinities of the following opioid peptides in competitionfor ³H-diprenorphine are set forth in FIG. 3: DADLE, a high affinityagonist for mu and delta receptors; DSLET and DPDPE, both high affinityagonists of delta (but not mu) receptors; DAGO, a selective agonist formu receptors; and Dynorphin 1-17, a high affinity agonist for kappareceptors and moderate to low affinity agonist for delta receptors. Asshown in FIG. 3, the displacement of ³H-diprenorphine, in decreasingorder of affinity, was observed for DSLET, DPDPE and DADLE, andDynorphin 1-17. Only weak displacement by DAGO was observed.

EXAMPLE 3 Northern Blot Analysis of RNA

[0148] For Northern analysis, the mRNA from NG108-15 cells, and fromcells dissected from regions of rat brain was separated byelectrophoresis through 2.2 M formaldehyde/1.5% agarose, blotted tonylon and hybridized in aqueous solution at high stringency. The filterswere prehybridized in 0.5 M NaPO₄, pH 7.2; 1% BSA; 1 mM EDTA; 7% SDS;and 100 μg/ml denatured salmon sperm DNA for at least four hours at 68°C. (Boulton et al., supra). The filters were then hybridized overnightunder these same conditions with a 5×10⁶ cpm/ml purified cDNA insertlabelled by random priming (A. P. Feinberg and B. Vogelstein, AnalBiochem (1983) 132:6). The filters were twice washed in 40 mM NaPO₄, pH7.2; 0.5% BSA; 5% SDS; and 1 mM EDTA for one hour, and then washed twicein 40 mM NaPO₄, pH 7.2; 1% SDS; and 1 mM EDTA for one hour each, all at68° C. Thereafter autoradiography was performed with DuPont CromexLightening Plus at −70° C.

[0149] The results of the Northern analysis of the mRNA showed thepresence of multiple bands hybridizing to the probe at approximately8.7, 6.8, 4.4, 2.75 and 2.2 kilobases (Kb) (FIG. 4). Also, the Northernanalysis indicates that the pattern of mRNA may vary between brainregions. At present, it is unclear whether these mRNAs encode differentprotein sequences, and if so, whether these messages represent differenttypes or sub-types of opioid receptors.

EXAMPLE 4 Southern Blot Analysis of DNA

[0150] The radiolabeled DOR-1 cDNA probe was hybridized to genomicSouthern blots by standard methods (Sambrook et al., supra).Accordingly, the radiolabeled DOR-1 cDNA probe was hybridized under highstringency conditions to a blot of NG108-15, mouse, rat and human DNAcut with restriction endonuclease BamHI (FIG. 7). Single bands wereobserved in the clones containing the NG108-15, mouse, and rat DNA. Thesizes of the bands hybridizing to the cDNA probe were estimated to be5.2 kb (NG108-15), 5.2 kb (mouse), and 5.7 kb (rat). These resultsindicate the close homology of the mouse and rat genes, and alsodemonstrate that the DOR-1 clone is from the murine parent of theNG108-15 cell line.

[0151] In a blot containing EcoRI-cut genomic DNA from many differentspecies, hybridization of the DOR-1 cDNA under conditions of moderatestringency showed two bands in each lane of mouse, rat, human, rabbit,and several other mammalian species. This demonstrates a closerelationship between opioid receptor genes in all of these species.Further, these results show that the genes or cDNAs from each of thesespecies may readily be cloned using hybridization under moderatestringency.

EXAMPLE 5 Determination of the cDNA Sequence

[0152] Isolated cDNA represented by the DOR clone was analyzed bysubcloning the insert from the cDNA clone into a plasmid such aspBluescript™ (Stratagene, San Diego, Calif.) and using the dideoxymethod (Sanger et al., Proc Natl Acad Sci USA (1977) 74:5463-5467). Thesequence of the cDNA was determined from single-stranded DNA andspecifically designed internal primers, using both Sequenase and ΔTaqcycle sequencing kits (USB). These kits, widely used in the art, utilizethe dideoxy chain termination method. The DNA sequence and predictedprotein sequence was then compared to sequences in established databankssuch as GenBank.

[0153] Sequencing the cDNA insert in the DOR-1 clone, revealed an openreading frame of 370 amino acids (FIG. 5). Comparisons with knownsequences in GenBank showed highest homology between DOR-1 and theG-protein-coupled somatostatin receptor (57% amino acid identity), andslightly lower homology with the receptors binding angiotensin, the twochemotactic factors IL-8 and N-formyl peptide. FIG. 6 shows the homologyto the human somatostatin 1 receptor. The close homology of the presentreceptor clone with the somatostatin receptor is especially noteworthysince somatostatin ligands are reported to bind to opioid receptors, andto have molecular mechanisms similar to those in delta receptors.

[0154] Other features of the DOR-1 clone amino acid sequence deducedfrom the cDNA sequence include three consensus glycosylation sites atresidues 18 and 33 (predicted to be in the extracellular N-terminaldomain), and at residue 310 (close to the C-terminus and predicted to beintracellular). Phosphokinase C consensus sites are present withinpredicted intracellular domains, at residues 242, 255, 344, and 352.Seven putative membrane-spanning regions were identified based onhydrophobicity profiles, as well as homology with Rhodopsin and otherG-protein coupled receptors which have been analyzed with respect tomembrane-spanning regions using MacVector (I.B.I.) analysis. The DOR-1clone isolated in accordance with the principles of the presentinvention produces a delta receptor with a predicted molecular weight of40,558 daltons prior to post-translational modifications such asN-glycosylation.

EXAMPLE 6 Isolation of Opioid Receptor Genomic Clones

[0155] Isolation of genomic clones was carried out according totechniques known in the art. To isolate opiate receptor genomic clones,300,000 human genomic clones in γgem 11 (Promega) and a similar numberof mouse genomic clones in lambda Fix (Stratagene) were plated on hoststrain Le392 and probed with the 1.1 kb DOR-1 Pst/Xba I fragment, whichcontains primarily the coding region. The conditions for hybridizationwere of fairly low stringency: 50% formamide/6× SSC, overnight at 37° C.The washes were performed also at low stringency: 2× SSC, 0.1% SDS atroom temperature.

[0156] One mouse clone and three human genomic clones were isolated andpurified by sequential rounds of hybridization and plaque purification.DNA preparation and restriction analysis showed that the three humanclones had very different EcoRI digestion patterns. The 1.1 kb opiatereceptor probe hybridized to a different single EcoRI band in Southernblot analysis for each clone. These results indicated preliminarily thatthree different genes were represented by the human genomic clones whichwere designated H3, H14 and H20 (see FIGS. 8a, 8 b, 8 c and 8 d). Eachof these clones was deposited on Aug. 13, 1993 at the American TypeCulture Collection, Rockville, Md., under conditions of the BudapestTreaty. All restrictions on access to these deposits will be irrevocablyremoved at the time a patent issues in the United States on the basis ofthis application. The ATCC deposit numbers are ______ for H3, ______ forH14, and ______ for H20.

[0157] The H3, H14 and H20 clones were digested into smaller fragmentsby EcoRI and TaqI and then shotgun cloned into the appropriate site ofBluescript for sequencing. The partial nucleotide sequence for H3 isshown in FIG. 8a; the partial nucleotide sequence of H14 is shown inFIG. 8b; the partial nucleotide sequence of H20 is shown in FIG. 8c.

[0158] The three genomic clones were mapped by in situ hybridization onhuman metaphase chromosomes by Dr. Glenn Evans of the Salk Institute. H3maps to chromosome 1P; H14 maps near the centromere of chromosome 8, andH20 maps to chromosome 6. Comparison of sequence data obtained asdescribed above with the published sequences for the murine counterpartsreferenced hereinabove, and with the DOR-2 clone described hereinbelow,confirmed that: (a) H3 encodes the human delta opioid receptor; (b) H14encodes the human kappa opioid receptor and (c) H20 encodes the human mureceptor. In addition, H20 appears to contain a CACACA marker (FIG. 8d)which provides a means to track the inheritance of this gene.

[0159] The genomic clones were digested into smaller fragments by EcoRIand TaqI, then shotgun cloned into the appropriate site of Bluescriptfor sequencing.

EXAMPLE 7 Isolation of Opioid Receptor Clones From Additional Organisms

[0160] In order to isolate the opioid receptor from mammalian braincells, for example human brain cells, a random-primed human brainstemcDNA library in λ Zap (Stratagene) was screened using the murine cDNAencoding the DOR-1 described herein. Positive plaques were purified andrescreened. Individual positive clones are sequenced and characterizedas above.

EXAMPLE 8 Determination of Probable Antigenic Sequences

[0161] By evaluating the amino acid sequence of the opicid receptorencoded by DOR-1 with the MacVector (I.B.I.) antigenic index, and theantigenic index in accordance to Jameson, B. and H. Wolf, Comput Applicin Biosci (1988) 4:181-186, the following underlined sequences of thedelta opioid receptor were determined to have a high antigenicpotential:

[0162] NH₂ MELVPSARAELOSSPLVNLSDAFPSAFPSAGANASGSPGARSASSLALAIAITALYSAVCAVGLLGNVLVMFGIVRYTKLKTATNIYIFNLALADALATSTLPFQSAKYLMETWPFGELLCKAVLSIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALDFRTPAKAKLINICIWVLASGVGVPIMVMAVTQPRDGAVVCMLQFPSPSWYWDTVTKICVFLFAFWPILIITVCYGLMLLRLRSVRLLSGSKEKDRSLRRITRMVLVVVGAFVVCWAPIHIFVIVWTLVDINRRDPLVVAALHLCIALGYANSSLNPVLYAFLDENFKRCFRQLCRTPCGROEPGSLRRPROATTRERVTACTPSDGPGGGAAA-COOH.

[0163] The N-terminal sequence is extracellular, the other foursequences are predicted to be intracellular.

EXAMPLE 9 Recovery of the Murine Clone DOR-2 (mMOR-1)

[0164] A cDNA library prepared from mouse brain in λgt10 was probedusing the low-stringency conditions of Example 6 using DOR-1 as a probe.One clone was recovered, inserted into Bluescript and sequenced.Northern and Southern blots indicated divergence from DOR-1. This clone,designated DOR-2, represented a new gene. DOR-2 hybridized to adifferent pattern of neurons than did DOR-1 and showed greater labelingof the striatum. Expression of DOR-1 by insertion into the vector pCDNAand transfection into mammalian cells produced cells which bindmorphine, indicative of a mu-receptor. The cells also bind thenonselective opiate antagonist diprenorphine. The identity of DOR-2(mMOR-1) as that of a mu receptor was confirmed by the displacement of³H-DPN by nanomolar concentrations of the mu-selective ligandsmorphlceptin, DAMGO and morphine. The delta selective ligands DPDPE anddeltorphan did not displace the binding and naloxone had the expectedhigh affinity. The partial sequence designated H20, described in Example6, was substantially similar to DOR-2. The partial sequence of DOR-2 isshown in FIG. 9.

[0165]FIG. 10 shows a comparison of the amino acid sequences of murinedelta receptin with the rat mu and kappa receptors. There are extensiveregions of homology.

EXAMPLE 10 Isolation of ORL-1

[0166] A human brain stem cDNA library was obtained from Stratagene andprobed using low-stringency hybridization with the murine DOR-l sequenceshown in FIG. 5 under stringency conditions of 50% formamide/6× SSC at37° C. with washes of 1× SSC/0.1 SDS at 37° C. A partial cDNA cloneencoding ORL-1 was obtained and completed at the 5′ end by RACE usingcDNA obtained from human brain. The DNA sequence obtained for ORL-1 isshown in FIG. 11 and is identical to that reported by Mollereau et al.(supra). ORL-1 has approximately 44% amino acid identity to the mureceptor.

[0167] In addition to ORL-1, three clones for ORL-2 were obtained and afull-length clone was assembled from two overlapping clones. Thesequence of one of the ORL-2 clones was identical to that reported byO'Dowd et al. (supra) while the other had a base change at Leu¹²⁹ whichdid not result in an alteration of amino acid sequence.

[0168]FIG. 12 compares the protein sequences of three cloned opioidreceptors and ORL-1 and ORL-2.

[0169] Multiple PKC and PKA cites in the third intracellular loop ofORL-1 are similar to those in the delta opioid receptor. However, a Hisresidue present in the sixth transmembrane domain of all the opioidreceptors is absent in ORL-1; this His residue may play a role inaromatic interaction with ligands and may be critical for opioidreceptor binding.

[0170] Mollereau et al. (supra) have shown that a stable cell linetransfected with ORL-1 shows etorphine-induced cyclase inhibition. Thisinhibition is reversible with diprenorphine, although labeleddiprenorphine binding to ORL-1 has not been shown. In addition., ORL-1has two Asn-linked glycosylation sites in the N-terminal extracellulardomain as shown in FIG. 12.

1. An isolated and purified or recombinant DNA molecule containing anucleotide sequence encoding an opioid receptor which hybridizes underconditions of low stringency to a probe consisting of the nucleotidesequence shown in FIG. 5 or to its complement.
 2. The DNA molecule ofclaim 1 wherein said nucleotide sequence encodes human delta opioidreceptor, human kappa opioid receptor, human mu opioid receptor, murinedelta opioid receptor, murine mu opioid receptor or ORL-1.
 3. A DNAmolecule comprising an expression system capable, when transformed intoa host, of producing an opioid receptor in the host, which expressionsystem comprises a nucleotide sequence encoding said opioid receptoroperably linked to heterologous control sequences operable in said host,wherein said opioid receptor is defined as encoded by a nucleotidesequence which hybridizes under conditions of low stringency to thenucleotide sequence of FIG. 5 or to its complement.
 4. The DNA moleculeof claim 3 wherein said opioid receptor is a human delta opioidreceptor, human kappa opioid receptor, human mu opioid receptor, murinedelta opioid receptor, murine mu opioid receptor, or ORL-1. 5.Recombinant host cells modified to contain the expression system ofclaim
 3. 6. A method to produce an opioid receptor protein which methodcomprises culturing the cells of claim 5 under conditions that effectexpression of the encoding DNA to produce said receptor protein, andrecovering the receptor protein from the culture.
 7. A method to producerecombinant cells that display opioid receptors at their surface, whichmethod comprises culturing the cells of claim 5 under conditions thateffect expression of the encoding DNA to produce said receptor proteinat their surface.
 8. Recombinant cells prepared by the method of claim7.
 9. A method to screen a candidate substance for opioid agonist orantagonist activity, which method comprises: incubating the cells ofclaim 8 in the presence and absence of the candidate substance underconditions suitable for detection of such activity, and detecting thepresence, absence or amount of said activity.
 10. An opioid receptorproduced by the method of claim
 6. 11. An antibody composition free ofred blood cells which comprises antibodies immunoreactive with theopioid receptor produced by the method of claim
 6. 12. A method fordetermining anatomical locations of opioid receptors in vertebrates,which method comprises: administering the antibody composition of claim11 to a vertebrate subject; waiting a sufficient time for said antibodycomposition to complex with said receptor; and detecting the location ofsaid complex in said subject.
 13. A method for blocking the interactionof opioids with opioid receptors which method comprises: contacting saidreceptors with the antibody composition of claim 11; and allowing saidcomposition to bind to said receptor.
 14. A method to modulate theexpression of DNA encoding an opioid receptor which method comprisestreating a cell capable of such expression with a DNA complementary tothe DNA of claim 1 under conditions wherein said DNA of claim 1hybridizes to said target DNA.